The primary goal of the present proposal is to better understand how Orb auto-regulates its own expression and how it controls the on site translation of mRNAs encoding factors critical for establishing oocyte/nurse cell identity and defining the polarity of the egg chamber and embryo. Part of the answer to these questions will come from identifying and characterizing other genes that are important for orb auto-regulation and/or function. In experiments that form the basis of the present application, we have used two complementary approaches to identify potential "co-factors." In the first we generated a sensitized orb mutant background which could be used to screen for second site mutations that "interact" genetically with orb. In the second, we used a biochemical approach to identify proteins that are associated with Orb in vivo. We uncovered many interesting "candidate" genes with these procedures during the previous grant period. We also characterized three of these "candidates", Squid (Sqd), Rasputin (Rin) and dFMR1 in some detail. Sqd and Rin function as positive regulators, promoting Orb expression/ activity, while dFMR1 is a negative regulator. During the upcoming grant period we propose to build on our previous studies, focusing on understanding how each of these proteins collaborates with other factors to regulate Orb expression/ activity. Our biochemical experiments suggest that Orb forms a series of distinct complexes. Since many of the factors found in these different Orb complexes were also identified independently in our genetic screen, we know that the Orb associated proteins are likely to be important for Orb function. We will focus our initial studies on factors associated with Orb:dFMR1 and(or) Orb:Rin complexes and will characterize these factors using approaches that were developed in earlier work on Sqd, Rin and dFMR1. Other interesting candidates will also be studied using the same procedures. To complement this analysis of Orb, we have begun analyzing the somatic Orb-like gene, orb-2.